Structure-Function Relationships Underlying the Capacity of Bordetella Adenylate Cyclase Toxin to Disarm Host Phagocytes

Abstract

Bordetellae, pathogenic to mammals, produce an immunomodulatory adenylate cyclase toxin-hemolysin (CyaA, ACT or AC-Hly) that enables them to overcome the innate immune defense of the host. CyaA subverts host phagocytic cells by an orchestrated action of its functional domains, where an extremely catalytically active adenylyl cyclase enzyme is delivered into phagocyte cytosol by a pore-forming repeat-in-toxin (RTX) cytolysin moiety. By targeting sentinel cells expressing the complement receptor 3, known as the CD11b/CD18 (α_Mβ₂) integrin, CyaA compromises the bactericidal functions of host phagocytes and supports infection of host airways by Bordetellae. Here, we review the state of knowledge on structural and functional aspects of CyaA toxin action, placing particular emphasis on signaling mechanisms by which the toxin-produced 3',5'-cyclic adenosine monophosphate (cAMP) subverts the physiology of phagocytic cells.

Keywords: Bordetella; CD11b/CD18; adenylate cyclase toxin; cAMP; cell signaling; complement receptor 3; innate immunity; membrane pores; repeats-in-toxin; β2 integrins.

Figure 1

Schematic representation of CyaA. CyaA is a 1706 residue-long polypeptide that consists of an N-terminal AC enzyme domain (~400 residues) and a C-terminal Hly moiety (~1300 residues) that are linked together with a ~100 residue-long segment (residues 400 to 500). The Hly portion of CyaA itself harbors several functional subdomains: (i) a hydrophobic pore-forming domain (residues 500 to 700); (ii) an activation domain (residues 800 and 1000), where the posttranslational acylation at two lysine residues (K860 and K983) occurs; (iii) a typical calcium-binding RTX domain with the nonapeptide repeats binding calcium ions and with the CD11b/CD18-binding segment (residues 1166–1287); and (iv) a C-terminal secretion signal.

Figure 2

The crystal structure of the AC domain of CyaA in the complex with the C-terminal domain of CaM [53]. α-helices and β-strands of the AC domain are colored in green and blue, respectively. The C-terminal domain of CaM is colored in red. Calcium and magnesium ions are represented by yellow and violet balls. The structure of the ATP analog adefovir diphosphate is represented by black lines.

Figure 3

The crystal structure of the segment 1529–1681 of CyaA [15]. The consecutive nonapeptide tandem repeats (G-G-X-G-X-D-X-X-X) are arranged in a regular right-handed helix of parallel β-strands (β-roll). β-strands and the C-terminal α-helix are colored in blue and green, respectively. Calcium ions are represented by yellow balls.

Figure 4

Schematic representation of the CD11b/CD18 heterodimer and its interaction with CyaA. (a) The 1152 residues long CD11b subunit contains an N-terminal secretion signal (16 residues; cyan), an N-terminal extracellular domain (1088 residues; yellow), a transmembrane segment (24 residues; brown) and a C-terminal cytoplasmic tail (24 residues; beige) [113,114]. The N-terminal part of CD11b consists of seven β-sheet repeats (1 to 7, from 52 to 79 residues; green), which have been predicted to fold into a β-propeller domain and a region of 187 residues, called the inserted or I-domain, that is localized between repeats 2 and 3 of the β-propeller (red) [115,116]. The I domain contains an Mg²⁺/Mn²⁺ coordination site at its surface (MIDAS) that is critical for ligand binding [115,117,118]. Repeats 5 to 7 of CD11b have EF hand-like Ca²⁺-binding motifs [116]. CyaA primarily recognizes the CD11b segment containing residues 614 to 682. The CD18 subunit is a 769 residues long polypeptide chain harboring an N-terminal secretion signal (22 residues; cyan), an N-terminal extracellular domain (678 residues; yellow), a transmembrane segment (23 residues; brown) and a C-terminal cytoplasmic domain (46 residues; beige) [119]. The extracellular domain contains the I-like domain (residues 124 to 363; red) and 56 cysteine residues forming the Cys-rich region localized near the membrane and composed of four repeat units of approximately 40 residues (green). These fold into small, very compact domains and are parts of the long stalk. The CD11b and CD18 subunits harbor several potential N-glycosylation sites (N-X-S/T), most of them modified by oligosaccharide chains linked to asparagine residues [120,121]. (b) 3D model of the CD11b subunit of CR3 (generated with the Modeler suite of programs) with the highlighted segment 614–682 (violet) that interacts with the segment 1166–1287 of CyaA (yellow; modeled using the structure prediction server I-TASSER) [18]. (c) The negatively charged residues of the 1166–1287 segment of CyaA are involved in the interaction with the positively charged and hydrophilic residues of the 614–682 segment of CD11b [18]. Reproduced and modified from [18], 2015, eLife Sciences Publications.

Figure 5

Schematic model of CyaA action on target membrane. After binding to CD11b/CD18 on myeloid cells [17,18], CyaA penetrates the cytoplasmic membrane and employs two distinct conformers to exert its multiple activities [68]. One would be the translocation precursor that would account for delivery of the invasive AC domain across the lipid bilayer [69] and provoke a concomitant influx of calcium ions into cell cytosol [28]. The other conformer would form a pore precursor that would oligomerize into CyaA pores [11,66,68], provoking potassium efflux from target cells [34]. Not drawn to scale.

Figure 6

Schematic depiction of CyaA interaction with target cells: cAMP-dependent signaling and its impact on epithelial cells and phagocytes. (a) CyaA toxin molecules secreted by the T1SS of Bordetella bind specifically to the integrin CD11b/CD18 (CR3, or α_Mβ₂) on the surface of phagocytes (monocytes, macrophages, DCs or neutrophils), or interact nonspecifically with the surfaces of other cell types, including epithelial cells. Following insertion into cytoplasmic membrane, CyaA translocates its enzymatic AC domain into the cytosol. In parallel, membrane-inserted CyaA molecules can oligomerize into cell-permeabilizing pores (not shown). Inside cell cytosol, the AC domain is activated by binding of calmodulin and catalyzes an extremely rapid and unregulated conversion of cytoplasmic ATP into cAMP, a key second messenger that hijacks the different cellular signaling pathways schematically delineated in the drawing (see the text for more detailed description of these pathways). The pointed arrowheads in the drawing indicate an activating effect and the flat arrowheads indicate an inhibitory effect under normal physiological conditions. The red color of the arrowhead indicates an inhibitory effect, or interference, elicited by the cAMP signaling of the CyaA toxin and the green color indicates an enhancing effect of CyaA/cAMP-triggered signaling action. (b) The outcomes of CyaA/cAMP signaling are shown together with the cell type for which they were originally described. Green color represents stimulation of particular interaction or process by CyaA action, while red color represents inhibition.